Restenoisis
Percutaneous transluminal coronary angioplasty (PTCA) is used to treat obstructive coronary artery disease by compressing atheromatous plaque to the sides of the vessel wall. PTCA is widely used with an initial success rate of over 90%. Approximately 666,000 angioplasties were conducted in the United States alone in 1996, and more of these procedures were performed on men (452,000) than women (214,000). Of this total, 482,000 were percutaneous transluminal coronary angioplasty (P.T.C.A. (American Heart Association; www.amhrt.org). Despite the frequent application of this procedure and its high initial success rate, the long-term success of PTCA is limited by intraluminal renarrowing or restenosis at the site of the procedure. This occurs within 6 months following the procedure in approximately 30% to 40% of patients who undergo a single vessel procedure and in more than 50% of those who undergo multivessel angioplasty.
Stent placement has largely supplanted balloon angioplasty because it is able to more widely restore intraluminal dimensions which has the effect of reducing restenosis by approximately 50%. Ironically, stent placement actually increases neointimal growth at the treatment site, but because a larger lumen can be achieved with stent placement, the tissue growth is more readily accommodate, and sufficient luminal dimensions are maintained, so that the restenosis rate is nearly halved by stent placement compared with balloon angioplasty alone.
The pathophysiological mechanisms involved in restenosis are not fully understood. While a number of clinical, anatomical and technical factors have been linked to the development of restenosis, at least 50% of the process has yet to be explained. However, it is known that following endothelial injury, a series of repair mechanisms are initiated. Within minutes of the injury, a layer of platelets and fibrin is deposited over the damaged endothelium. Within hours to days, inflammatory cells begin to infiltrate the injured area. Within 24 hours after an injury, vascular smooth muscle cells (SMCs) located in the vessel media commence DNA synthesis. A few days later, these activated, synthetic SMCs migrate through the internal elastic lamina towards the luminal surface. A neointima is formed by these cells by their continued replication and their production of extracellular matrix. An increase in the intimal thickness occurs with ongoing cellular proliferation matrix deposition. When these processes of vascular healing progress excessively, the pathological condition is termed intimal hyperplasia or neointimial hyperplasia. Histological studies in animal models have identified neointimal hyperplasia as the central element in restenosis.
Neointimal hyperplasia is understood to figure prominently in peripheral vascular restenosis following reconstructive procedures. One series of 5,000 arterial reconstructions reports 50% of late failures to be due to neointimal hyperplasia (Imparato et al. (1972) Surg. 72:1107-1117). Restenosis following stenting is similarly thought to involve an important component of neointimal hyperplasia (Dussaillant et al. (1995) J. Am. Coll. Cardiol 26:720-724). In the coronary system, by contrast, restenosis following balloon angioplasty involves vascular remodeling as well as neointimal hyperplasia. The importance of vascular remodeling in this setting may be attributable to the nature of the injury to the vessel wall following balloon angioplasty. Commonly, the injury to the vessel wall with this procedure involves dissection planes extending through the atherosclerotic plaque into the vessel media (Mintz et al. (1996) Circ. 94:35043). Furthermore, plaque fracture, medial stretch, focal medial rupture and adventitial stretch all may occur following angioplasty. Repair of the deeper layers of the vessel wall takes place by the general processes of wound healing, including inflammation, neovascularization, fibroblast proliferation and eventual collagen deposition. Cumulatively, these processes lead to remodeling of the coronary vessel wall that may culminate in restenosis.
The biology of vascular wall healing implicated in restenosis therefore includes the general processes of wound healing and the specific processes of neointimal hyperplasia. Inflammation is generally regarded as an important component in both these processes. (Munro and Cotran (1993) Lab. Investig. 58:249-261; and Badimon et al. (1993), Supp II 87:3-6). Understanding the effects of acute and chronic inflammation in the blood vessel wall can thus suggest methods for diagnosing and treating restenosis and related conditions.
In its initial phase, inflammation is characterized by the adherence of leukocytes to the vessel wall. Leukocyte adhesion to the surface of damaged endothelium is mediated by several complex glycoproteins on the endothelial and neutrophil surfaces. Two of these binding molecules have been well-characterized: the endothelial leukocyte adhesion molecule-1 (ELAM-1) and the intercellular adhesion molecule-1 (ICAM-1). During inflammatory states, the attachment of neutrophils to the involved cell surfaces is greatly increased, primarily due to the upregulation and enhanced expression of these binding molecules. Substances thought to be primary mediators of the inflammatory response to tissue injury, including interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-xcex1), lymphotoxin and bacterial endotoxins, all increase the production of these binding substances.
After binding to the damaged vessel wall, leukocytes migrate into it. Once in place within the vessel wall, the leukocytes, in particular activated macrophages, then release additional inflammatory mediators, including IL-1, TNF, prostaglandin E2, (PGE2), bFGF, and transforming growth factors xcex1 and xcex2 (TGFxcex1, TGFxcex2). All of these inflammatory mediators recruit more inflammatory cells to the damaged area, and regulate the further proliferation and migration of smooth muscle. A well-known growth factor elaborated by the monocyte-macrophage is monocyte- and macrophage-derived growth factor (MDGF), a stimulant of smooth muscle cell and fibroblast proliferation. MDGF is understood to be similar to platelet-derived growth factor (PDGF); in fact, the two substances may be identical. By stimulating smooth muscle cell proliferation, inflammation can contribute to the development and the progression of neointimal hyperplasia.
Leukocytes, attracted to the vessel wall by the abovementioned chemical mediators of inflammation, produce substances that have direct effects on the vessel wall that may exacerbate the local injury and prolong the healing response. First, leukocytes activated by the processes of inflammation secrete lysosomal enzymes that can digest collagen and other structural proteins. Releasing these enzymes within the vessel wall can affect the integrity of its extracellular matrix, permitting SMCs and other migratory cells to pass through the wall more readily. Hence, the release of these lysosomal proteases can enhance the processes leading to neointimal hyperplasia Second, activated leukocytes produce free radicals by the action of the NADPH system on their cell membranes. These free radicals can damage cellular elements directly, leading to an extension of a local injury or a prolongation of the cycle of injury-inflammation-healing.
The responses to vascular injury that lead to restenosis have certain features in common with the processes leading to the development of the vascular lesions of atherosclerosis. Currently, it is understood that the lesions of atherosclerosis are initiated by some form of injury to arterial endothelium, whether due to hemodynamic factors, endothelial dysfunction or a combination of these or other factors (Schoen, xe2x80x9cBlood vessels, xe2x80x9dpp. 467-516 in Pathological Basis of Disease (Philadelphia: Saunders, 1994)). Inflammation has been implicated in the formation and progression of atherosclerotic lesions. Several inflammatory products, including IL-1xcex2, have been identified in atherosclerotic lesions or in the endothelium of diseased coronary arteries (Galea, et al. (1996) Arterioscler Thromb Vasc Biol. 16:1000-6). Also, serum concentrations of IL-1xcex2 are elevated in patients with coronary disease (Hasdai, et al. (1996) Heart, 76:24-8). Realizing the importance of inflammatory processes in the final common pathways of vascular response to injury allows analogies to be drawn between the lesions seen in restenosis and those seen in atherosclerosis.
Currently, approximately 500,000 patients per year undergo vascular reconstructive procedures, with half involving the coronary vessels and the other half involving the periphery. Restenosis and progressive atherosclerosis are the most common mechanisms for late failure in these reconstructions. It would be desirable to determine which patients would respond well to invasive treatments for occlusive vascular disease such as angioplasty and intravascular stent placement. It would be further desirable to identify those patients at increased risk for stenosis so that they could be targeted with appropriate therapies to prevent, modulate or reverse the condition. It would be desirable, moreover, to identify those individuals for whom PTCA and stent placement is a suboptimal therapeutic choice because of the risk of restenosis. Those patients might become candidates at earlier stages for vascular reconstructive procedures, possibly combined with other pharmacological interventions.
Genetics of the IL-1 Gene Cluster
The IL-1 gene cluster is on the long arm of chromosome 2 (2q13) and contains at least the genes for IL-1xcex1 (IL-1A), IL-1xcex2 (IL-1B), and the IL-1 receptor antagonist (IL-1RN), within a region of 430 Kb (Nicklin, et al. (1994) Genomics, 19: 382-4). The agonist molecules, IL-1xcex1 and IL-1xcex2, have potent pro-inflammatory activity and are at the head of many inflammatory cascades. Their actions, often via the induction of other cytokines such as IL-6 and IL-8, lead to activation and recruitment of leukocytes into damaged tissue, local production of vasoactive agents, fever response in the brain and hepatic acute phase response. All three IL-1 molecules bind to type I and to type II IL-1 receptors, but only the type I receptor transduces a signal to the interior of the cell. In contrast, the type II receptor is shed from the cell membrane and acts as a decoy receptor. The receptor antagonist and the type II receptor, therefore, are both anti-inflammatory in their actions.
Inappropriate production of IL-1 plays a central role in the pathology of many autoimmune and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disorder, psoriasis, and the like. In addition, there are stable inter-individual differences in the rates of production of IL-1, and some of this variation may be accounted for by genetic differences at IL-1 gene loci. Thus, the IL-1 genes are reasonable candidates for determining part of the genetic susceptibility to inflammatory diseases, most of which have a multifactorial etiology with a polygenic component. Indeed, there is increasing evidence that certain alleles of the IL-1 genes are over-represented in these diseases.
Certain alleles from the IL-1 gene cluster are already known to be associated with particular disease states. For example, IL-1RN allele 2 has been shown to be associated with coronary artery disease (PCT/US/98/04725, and U.S. Ser. No. 08/813456), osteoporosis (U.S. Pat. No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al. (1996) Hum. Genet 97(3): 369-74), alopecia areata (Cork, et al., (1995) J. Invest. Dermatol. 104(5 Supp.): 15S-16S; Cork et al. (1996) Dermatol Clin 14: 671-8), Graves disease (Blakemore, et al. (1995) J. Clin. Endocrinol. 80(1): 111-5), systemic lupus erythematosus (Blakemore, et al. (1994) Arthritis Rheum. 37: 1380-85), lichen sclerosis (Clay, et al. (1994) Hum. Genet. 94: 407-10), and ulcerative colitis (Mansfield, et al. (1994) Gastoenterol. 106(3): 637-42).
In addition, the IL-1A allele 2 from marker xe2x88x92889 and IL-1B (TaqI) allele 2 from marker +3954 have been found to be associated with periodontal disease (U.S. Pat. No. 5,686,246; Kormman and diGiovine (1998) Ann Periodont 3: 327-38; Hart and Komman (1997) Periodontol 2000 14: 202-15; Newman (1997) Compend Contin Educ Dent 18: 881 -4; Komman et al. (1997) J. Clin Periodontol 24: 72-77). The IL-1A allele 2 from marker xe2x88x92889 has also been found to be associated with juvenile chronic arthritis, particularly chronic iridocyclitis (McDowell, et al. (1995) Arthritis Rheum. 38: 221-28 ). The IL-1B (TaqI) allele 2 from marker +3954 of IL-1B has also been found to be associated with psoriasis and insulin dependent diabetes in DR3/4 patients (di Giovine, et al. (1995) Cytokine 7: 606; Pociot, et al. (1992) Eur J. Clin. Invest. 22: 396-402). Additionally, the IL-1RN (VNTR) allele 1 has been found to be associated with diabetic retinopathy (see U.S. Ser No. 09/037472, and PCT/GB97/02790). Furthermore allele 2 of IL-1RN (VNTR) has been found to be associated with ulcerative colitis in Caucasian populations from North America and Europe (Mansfield, J. et al., (1994) Gastroenterology 106: 637-42). Interestingly, this association is particularly strong within populations of ethnically related Ashkenazi Jews (PCT W097/25445).
Genotype Screening
Traditional methods for the screening of heritable diseases have depended on either the identification of abnormal gene products (e.g., sickle cell anemia) or an abnormal phenotype (e.g., mental retardation). These methods are of limited utility for heritable diseases with late onset and no easily identifiable phenotypes such as, for example, a predisposition to restenosis. With the development of simple and inexpensive genetic screening methodology, it is now possible to identify polymorphisms that indicate a propensity to develop disease, even when the disease is of polygenic origin. The number of diseases that can be screened by molecular biological methods continues to grow with increased understanding of the genetic basis of multifactorial disorders.
Genetic screening (also called genotyping or molecular screening), can be broadly defined as testing to determine if a patient has mutations (or alleles or polymorphisms) that either cause a disease state or are xe2x80x9clinkedxe2x80x9d to the mutation causing a disease state. Linkage refers to the phenomenon that DNA sequences which are close together in the genome have a tendency to be inherited together. Two sequences may be linked because of some selective advantage of co-inheritance. More typically, however, two polymorphic sequences are co-inherited because of the relative infrequency with which meiotic recombination events occur within the region between the two polymorphisms. The co-inherited polymorphic alleles are said to be in linkage disequilibrium with one another because, in a given human population, they tend to either both occur together or else not occur at all in any particular member of the population. Indeed, where multiple polymorphisms in a given chromosomal region are found to be in linkage disequilibrium with one another, they define a quasi-stable genetic xe2x80x9chaplotype.xe2x80x9d In contrast, recombination events occurring between two polymorphic loci cause them to become separated onto distinct homologous chromosomes. If meiotic recombination between two physically linked polymorphisms occurs frequently enough, the two polymorphisms will appear to segregate independently and are said to be in linkage equilibrium.
While the frequency of meiotic recombination between two markers is generally proportional to the physical distance between them on the chromosome, the occurrence of xe2x80x9chot spotsxe2x80x9d as well as regions of repressed chromosomal recombination can result in discrepancies between the physical and recombinational distance between two markers. Thus, in certain chromosomal regions, multiple polymorphic loci spanning a broad chromosomal domain may be in linkage disequilibrium with one another, and thereby define a broad-spanning genetic haplotype. Furthermore, where a disease-causing mutation is found within or in linkage with this haplotype, one or more polymorphic alleles of the haplotype can be used as a diagnostic or prognostic indicator of the likelihood of developing the disease. This association between otherwise benign polymorphisms and a disease-causing polymorphism occurs if the disease mutation arose in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events. Therefore identification of a human haplotype which spans or is linked to a disease-causing mutational change, serves as a predictive measure of an individual""s likelihood of having inherited that disease-causing mutation. Importantly, such prognostic or diagnostic procedures can be utilized without necessitating the identification and isolation of the actual disease-causing lesion. This is significant because the precise determination of the molecular defect involved in a disease process can be difficult and laborious, especially in the case of multifactorial diseases such as inflammatory disorders.
Indeed, the statistical correlation between an inflammatory disorder and an IL-1 polymorphism does not necessarily indicate that the polymorphism directly causes the disorder. Rather the correlated polymorphism may be a benign allelic variant which is linked to (i.e. in linkage disequilibrium with) a disorder-causing mutation which has occurred in the recent human evolutionary past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the intervening chromosomal segment. Thus, for the purposes of diagnostic and prognostic assays for a particular disease, detection of a polymorphic allele associated with that disease can be utilized without consideration of whether the polymorphism is directly involved in the etiology of the disease. Furthermore, where a given benign polymorphic locus is in linkage disequilibrium with an apparent disease-causing polymorphic locus, still other polymorphic loci which are in linkage disequilibrium with the benign polymorphic locus are also likely to be in linkage disequilibrium with the disease-causing polymorphic locus. Thus these other polymorphic loci will also be prognostic or diagnostic of the likelihood of having inherited the disease-causing polymorphic locus. Indeed, a broad-spanning human haplotype (describing the typical pattern of co-inheritance of alleles of a set of linked polymorphic markers) can be targeted for diagnostic purposes once an association has been drawn between a particular disease or condition and a corresponding human haplotype. Thus, the determination of an individual""s likelihood for developing a particular disease of condition can be made by characterizing one or more disease-associated polymorphic alleles (or even one or more disease-associated haplotypes) without necessarily determining or characterizing the causative genetic variation.
In one aspect, the present invention provides novel methods and kits for determining whether a subject has or is predisposed to developing restenosis. Diagnosis of the presence of a restenosis disorder identifies those patients predisposed to the development of a restenosis disease, characterized by clinical events related to the recurrence of the initial vascular stenosis that is being treated by the stent. Determining which patients are at risk for developing the disease because they have the disorder thus opens the possibility of selecting therapies for the initial vascular stenosis most likely to avoid subsequent stenoses. Such patients might be preferred candidates for surgical revascularization rather than percutaneous transluminal angioplasty, for example, or such patients may benefit from pharmacological or topical interventions at an early stage that could affect the progression of the restenosis disorder.
In one embodiment, the method comprises determining whether a restenosis associated allele is present in a nucleic acid sample obtained from the subject., In a preferred embodiment, the restenosis associated allele is selected from the group consisting of allele 1 of each of the following markers: IL-1A (+4845), IL-1B(+3954), IL-1B(xe2x88x92511), IL-1RN (+2018) and IL-1RN (VNTR) or an allele that is in linkage disequilibrium with one of the aforementioned alleles. In preferred embodiments, the presence of a particular allelic pattern of one or more of the abovementioned IL-1 polymorphic loci is used to predict the susceptibility of an individual to developing restenosis. In particular, there are three patterns of alleles at four polymorphic loci in the IL-1 gene cluster that show various associations with particular cardiovascular disorders. These patterns are referred to herein as patterns 1, 2 and 3. Pattern 1 comprises an allelic pattern including allele 2 of IL-1A (+4845) or IL-1B(+3954) and allele 1 of IL-1B(xe2x88x92511) or IL-1RN (+2018), or an allele that is in linkage disequilibrium with one of the aforementioned allele. In a preferred embodiment, this allelic pattern permits the diagnosis of occlusive cardiovascular disorder. Pattern 2 comprises an allelic pattern including allele 2 of IL-1B (xe2x88x92511) or IL-1RN (+2018) and allele 1 of IL-1A (+4845) or IL-1B (+3954), or an allele that is in linkage disequilibrium with one of the aforementioned alleles. In a preferred embodiment, this allelic pattern permits the diagnosis of occlusive cardiovascular disorder. Pattern 3 comprises an allelic pattern including allele 1 of IL-1A (+4845) or allele 1 of IL-1B (+3954), and allele 1 of IL-1B (xe2x88x92511) or allele 1 of IL-1RN (+2018), or an allele that is in linkage disequilibrium with one of the aforementioned alleles. In a preferred embodiment, this allelic pattern permits the diagnosis of a restenosis disorder.
In another embodiment, the method of the invention may be employed by detecting the presence of an IL-1 associated polymorphism that is in linkage disequilibrium with one or more of the aforementioned restenosis-predictive alleles. For example, the following alleles of the IL-1 (44112332) haplotype are known to be in linkage disequilibrium:
Also, the following alleles of the IL-1 (33221461) haplotype are in linkage disequilibrium:
A restenosis associated allele can be detected by any of a variety of available techniques, including: 1) performing a hybridization reaction between a nucleic acid sample and a probe that is capable of hybridizing to the allele; 2) sequencing at least a portion of the allele; or 3) determining the electrophoretic mobility of the allele or fragments thereof (e.g., fragments generated by endonuclease digestion). The allele can optionally be subjected to an amplification step prior to performance of the detection step. Preferred amplification methods are selected from the group consisting of: the polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), cloning, and variations of the above (e.g. RT-PCR and allele specific amplification). Oligonucleotides necessary for amplification may be selected for example, from within the IL-1 gene loci, either flanking the marker of interest (as required for PCR amplification) or directly overlapping the marker (as in ASO hybridization). In a particularly preferred embodiment, the sample is hybridized with a set of primers, which hybridize 5xe2x80x2 and 3xe2x80x2 in a sense or antisense sequence to the restenosis associated allele, and is subjected to a PCR amplification.
A restenosis associated allele may also be detected indirectly, e.g. by analyzing the protein product encoded by the DNA. For example, where the marker in question results in the translation of a mutant protein, the protein can be detected by any of a variety of protein detection methods. Such methods include immunodetection and biochemical tests, such as size fractionation, where the protein has a change in apparent molecular weight either through truncation, elongation, altered folding or altered post-translational modifications.
In another aspect, the invention features kits for performing the above-described assays. The kit can include a nucleic acid sample collection means and a means for determining whether a subject carries a restenosis associated allele. The kit may also contain a control sample either positive or negative or a standard and/or an algorithmic device for assessing the results and additional reagents and components including: DNA amplification reagents, DNA polymerase, nucleic acid amplification reagents, restrictive enzymes, buffers, a nucleic acid sampling device, DNA purification device, deoxynucleotides, oligonucleotides (e.g. probes and primers) etc.
As described above, the control samples may be positive or negative controls. Further, the control sample may contain the positive (or negative) products of the allele detection technique employed. For example, where the allele detection technique is PCR amplification, followed by size fractionation, the control sample may comprise DNA fragments of the appropriate size. Likewise, where the allele detection technique involves detection of a mutated protein, the control sample may comprise a sample of mutated protein. However, it is preferred that the control sample comprises the material to be tested. For example, the controls may be a sample of genomic DNA or a cloned portion of the IL-1 gene cluster. Preferably, however, the control sample is a highly purified sample of genomic DNA where the sample to be tested is genomic DNA.
The oligonucleotides present in said kit may be used for PCR amplification of the region of interest or for direct allele specific oligonucleotide (ASO) hybridization to the markers in question. Thus, the oligonucleotides may either flank the marker of interest (as required for PCR amplification) or directly overlap the marker (as in ASO hybridization).
Such oligonucleotides can include, but are not limited to:
5xe2x80x2 ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3xe2x80x2 (SEQ ID No. 1) and 5xe2x80x2 AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3xe2x80x2 (SEQ ID No. 2) which can be used to amplify the human IL-1A (+4845) polymorphic locus;
5xe2x80x2 TGG CAT TGA TCT GGT TCA TC 3xe2x80x2 (SEQ ID No. 3) and 5xe2x80x2 GTT TAG GAA TCT TCC CAC TT-3xe2x80x2 (SEQ ID No. 4) which can be used to amplify the human IL-1B (xe2x88x92511) polymorphic locus;
5xe2x80x2-CTC AGG TGT CCT CGA AGA AAT CAA A-3xe2x80x2 (SEQ ID No. 5) and 5xe2x80x2 GCT TTT TTG CTG TGA GTC CCG-3xe2x80x2 (SEQ ID No. 6) which can be used to amplify the human IL-1B (+3954) polymorphic locus;
5xe2x80x2-CTC.AGC.AAC.ACT.CCT.AT-3xe2x80x2 (SEQ ID NO. 7) and 5xe2x80x2-TCC.TGG.TCT.GCA.GCT.AA-3xe2x80x2 (SEQ ID NO. 8) which can be used to amplify the human IL-1RN (VNTR) polymorphic locus;
5xe2x80x2-CTA TCT GAG GAA CAA CCA ACT AGT AGC-3xe2x80x2 (SEQ ID NO. 9) and 5xe2x80x2-TAG GAC ATT GCA CCT AGG GTT TGT-3xe2x80x2 (SEQ ID NO. 10) which can be used to amplify the human IL-1RN (+2018) polymorphic locus;
5xe2x80x2 ATT TTT TTA TAA ATC ATC AAG CCT AGG GCA 3xe2x80x2 (SEQ. ID No. 11) and 5xe2x80x2 AAT TAA AGG AGG GAA GAA TGA CAG AAA TGT 3xe2x80x2 (SEQ. ID No. 12) which can also be used to amplify the human IL-1A (+4845) polymorphic locus;
5xe2x80x2-AAG CTT GTT CTA CCA CCT GAA CTA GGC.-3xe2x80x2 (SEQ. ID NO. 13) and 5xe2x80x2-TTA CAT ATG AGC CTT CCA TG.-3xe2x80x2 (SEQ. ID NO. 14) which can be used to amplify the human IL-1A (xe2x88x92889) polymorphic locus;
Information obtained using the assays and kits described herein (alone or in conjunction with information on another genetic defect or environmental factor, which contributes to restenosis) is useful for determining whether a non-symptomatic subject has or is likely to develop restenosis. In addition, the information can allow a more customized approach to preventing the onset or progression of restenosis. For example, this information can enable a clinician to more effectively prescribe a therapy that will address the molecular basis of restenosis. In yet a further aspect, the invention features methods for treating or preventing the development of restenosis in a subject by administering to the subject an appropriate restenosis therapeutic of the invention. In still another aspect, the invention provides in vitro or in vivo assays for screening test compounds to identify restenosis therapeutics. In one embodiment, the assay comprises contacting a cell transfected with a restenosis causative mutation that is operably linked to an appropriate promoter with a test compound and determining the level of expression of a protein in the cell in the presence and in the absence of the test compound. In a preferred embodiment, the restenosis causative mutation results in decreased production of IL-1 receptor antagonist, and increased production of the IL-1 receptor antagonist in the presence of the test compound indicates that the compound is an agonist of IL-1 receptor antagonist activity. In another preferred embodiment, the restenosis causative mutation results in increased production of IL-1xcex1 or IL-1xcex2, and decreased production of IL-1xcex1 or IL-1xcex2 in the presence of the test compound indicates that the compound is an antagonist of IL-1xcex1 or IL-1xcex2 activity. In another embodiment, the invention features transgenic non-human animals and their use in identifying antagonists of IL-1xcex1 or IL-1xcex2 activity or agonists of IL-1Ra activity.
Other embodiments and advantages of the invention are set forth in part in the description which follows, and will be obvious from this description.